Application of pear source PcMIR397b promoter in construction of pear instantaneous transformation vector

Abstract

The invention belongs to the technical field of molecular biology, and particularly discloses application of a pear source PcMIR397b promoter in construction of a pear instantaneous transformation vector. The pear source PcMIR397b promoter which is high in expression efficiency and wide in tissue compatibility is screened and identified, the capacity of the promoter for driving target gene expression in a pear isolated organ is remarkably superior to that of a common heterologous promoter and a common endogenous promoter, and the pear source PcMIR397b promoter can efficiently play a role in different parts such as stems and leaves. The invention also provides an instantaneous transformation method of a pear plant, which combines the endogenous efficient pear source PcMIR397b promoter with a traditional agrobacterium-mediated transformation process, breaks through the limitation of application of a heterologous promoter in pears, and constructs an instantaneous transformation system which is more adaptive to pear species, convenient to popularize and use, rapid and efficient. The method is suitable for rapid verification of pear gene functions, and provides key technical support for improvement of pear gene editing efficiency and acceleration of a high-quality breeding process.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology and relates to pear-derived substances. PcMIR397b Application of promoters in constructing pear transient transformation vectors. Background Technology

[0002] A promoter is a DNA sequence that RNA polymerase recognizes, binds to, and initiates transcription. It contains conserved sequences required for RNA polymerase-specific binding and transcription initiation, and is mostly located upstream of the transcription start site of structural genes. The promoter itself is not transcribed. In gene expression, the promoter is one of the most important components. Based on their origin, promoters are divided into exogenous promoters derived from outside the host and endogenous promoters derived from the host itself. Currently, the promoters used in pear transgenic gene expression are mainly exogenous promoters. However, because exogenous promoters are not native to the host, they often exhibit low efficiency when used for gene expression (such as transient expression or stable transgenesis), and are prone to loss or methylation after long passage times.

[0003] Agrobacterium-mediated transient transformation is an important method for rapid gene function identification and is widely used in plant science research. Its technical system is relatively mature in model plants such as tobacco and tomato. However, due to limitations in transformation efficiency, many crops still lack fully mature transient transformation methods. Traditional transient transformation systems in pear plants exhibit low expression efficiency, which cannot meet the needs of gene function verification. Currently, endogenous strong promoters suitable for pear plant transient transformation are rarely reported. Therefore, developing suitable endogenous strong promoters for pear transient transformation vectors or pear plant transient transformation methods is of great significance for further development of pear gene editing and breeding. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a pear source. PcMIR397b Application of promoters in constructing pear transient transformation vectors. This invention creatively discovers the pear source. PcMIR397b The promoter can be effectively adapted to the construction of pear transient transformation vectors and the transient transformation process of pear plants. Its core advantage lies in its ability to drive the target gene to achieve efficient transient expression in pear plants and its broad tissue compatibility, providing a stable, reliable and efficient technical approach for the rapid verification of pear gene function.

[0005] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions: Firstly, this invention provides a pear source. PcMIR397b Application of promoters in constructing pear transient transformation vectors. This invention creatively screens and identifies a pear-derived gene that achieves a dual breakthrough in expression efficiency and tissue compatibility. PcMIR397bThis promoter demonstrates a significantly superior ability to drive target gene expression in isolated pear organs compared to commonly used heterologous and endogenous promoters. Furthermore, it exhibits broad tissue compatibility, functioning efficiently in various locations such as stems and leaves. (Pear source) PcMIR397b Promoters can provide more suitable expression regulatory elements for the rapid verification of pear gene function, and provide key technical support for improving the efficiency of pear gene editing and accelerating the process of high-quality breeding.

[0006] From the perspective of expressive efficiency, it includes pear source PcMIR397b The pear transient transformation vector with the promoter can drive the target gene to be expressed efficiently in pear plants, and the results show that it is significantly superior to commonly used heterologous vectors. CaMV35S Promoter, Pear Source pUBQq Promoter, Pear Source PcMIR397a Promoters such as the pear-derived pU6 promoter address the problem of insufficient expression efficiency of traditional promoters in pears; in terms of applicability, it includes pear-derived... PcMIR397b The pear transient transformation vector of the promoter has broad tissue compatibility in expression, and can be expressed in different parts such as stems and leaves, without tissue specificity, which can meet the needs of multi-organ gene function verification.

[0007] Preferably, the pear source PcMIR397b The nucleotide sequence of the promoter is shown in SEQ ID NO.1.

[0008] Secondly, the present invention provides a pear transient conversion carrier, wherein the pear transient conversion carrier comprises the aforementioned pear source. PcMIR397b A recombinant vector for a promoter gene; the recombinant vector includes the target gene.

[0009] Preferably, the recombinant vector uses a plant expression vector as its backbone; The plant expression vector includes at least one of the following: pCAMBIA series vectors, pBI221 vector, pBI121 vector, pBI121-EGFP vector, pBI121-mCherry or pUBI10-GUS vector.

[0010] Preferably, the target gene includes a reporter gene or a foreign gene in the vector backbone.

[0011] For example, using the pBI121 vector as a backbone, pear-derived... PcMIR397b The promoter gene replaces the one inherent in the pBI121 vector. CaMV35S Promoter genes were used to construct transient transformation vectors for pear. Furthermore, based on this, the full-length CDS sequence of the exogenous target gene can be used to replace the inherent GUS gene in the pBI121 vector to construct vectors containing specific exogenous target genes and pear-derived... PcMIR397b Pear transient conversion carrier of promoter.

[0012] Thirdly, the present invention also provides a method for the instantaneous transformation of pear plants, comprising the following steps: S1. Construct the pear instantaneous transformation carrier described in the second aspect; S2. The pear transient transformation vector is transformed into Agrobacterium to obtain a recombinant strain; S3. After being subjected to graded expansion culture and induction culture, the recombinant strain was mixed with pear explants and vacuum-permeable to obtain pear explants infected with the recombinant strain; S4. After culturing pear explants infected with the recombinant strain, the expression of the target gene is measured.

[0013] The instantaneous transformation method for pear plants provided by this invention combines an endogenous high-efficiency promoter with the traditional Agrobacterium-mediated transformation process, breaking through the limitations of heterologous promoters in pears and constructing a more suitable, efficient instantaneous transformation system that is easy to promote and use.

[0014] This invention reduces experimental errors through standardized operations such as graded expansion culture and induced culture, and ensures the repeatability and accuracy of target gene expression detection results by combining controllable culture conditions, thus providing a reliable basis for gene function verification.

[0015] Preferably, the pear explants include leaves from pear detached plants or tissue culture seedlings.

[0016] Preferably, in step S4, the culture includes dark culture and / or photoperiodic culture.

[0017] More preferably, the dark culture temperature is 23℃-27℃ and the culture time is 6h-18h.

[0018] More preferably, the light cycle is set to a light duration of 10h-18h and a darkness duration of 6h-14h.

[0019] More preferably, the photoperiodic culture temperature is 23℃-27℃ and the culture time is 24h-72h.

[0020] The instantaneous transformation method for pear plants provided by this invention has a wide range of applications. Combined with the environmental control of dark culture and photoperiodic culture, it can meet the transformation needs of various pear tissues, including pear in vitro plants or tissue culture seedling leaves.

[0021] More preferably, this invention uses the pBI121 vector as a backbone to construct the pear transient transformation vector p397b-pBI121, providing a method for transient transformation of pear plants. The transformation method includes the following steps: S1. Using the pBI121 vector as a backbone, the nucleotide sequence of the pear-derived vector is as shown in SEQ ID NO.1. PcMIR397bThe promoter gene sequence is replaced in the pBI121 vector. CaMV35S The gene sequence of the promoter was obtained from the pear transient transformation vector p397b-pBI121; S2. The pear transient transformation vector p397b-pBI121 was transformed into Agrobacterium to obtain a recombinant strain; S3. After secondary expansion culture and induction culture, the recombinant strain is mixed with leaves of pear in vitro plants or tissue culture seedlings, and after adding surfactant, it is vacuum-permeated to obtain pear explants infected with the recombinant strain. S4. After the pear explants infected with the recombinant strain were cultured in the dark and under photoperiod, the expression of the GUS gene was measured.

[0022] For example, the culture medium used for dark culture is MS solid medium.

[0023] More preferably, the method for constructing pear explants infected with the recombinant strain includes the following steps: S31. The recombinant strain was inoculated into YEB medium containing rifampicin and kanamycin and cultured at 25℃-30℃ and 160rpm-200rpm for 14h-18h to obtain the primary expansion medium; S32. The primary expansion medium is inoculated into YEB medium containing rifampicin, kanamycin, MES buffer and acetylsuccinone (AS), and cultured at 25℃-30℃ and 160rpm-200rpm for 4h-6h to obtain the secondary expansion medium; S33. Collect the bacterial cells in the secondary expansion broth, add induction solution to resuspend the bacterial cells, and incubate at 25℃-30℃ for 2h-4h to obtain the induced recombinant bacterial solution; S34. The induced recombinant bacterial solution is mixed with pear explants or tissue culture seedlings after subculture, an organosilicon surfactant is added, and the mixture is vacuum-permeated to obtain pear explants infected with the recombinant bacterial strain.

[0024] For example, the silicone surfactant includes silwet L-77. For example, step S34 specifically includes taking out pear explants or leaves from tissue culture seedlings that have been subcultured for 3 weeks, placing the plant or leaves in the induced recombinant bacterial solution, adding 0.01% silwet L-77, placing it in a vacuum pump, and performing vacuum permeation infection twice at a vacuum degree of -0.09 MPa, each time for 5 minutes; after the vacuum permeation infection is completed, pouring out the bacterial solution, absorbing the excess bacterial solution on the plant or leaves, and obtaining pear explants infected with the recombinant strain.

[0025] This invention modifies the pBI121 vector to deliver pear-derived... PcMIR397b The promoter replaces the inherent promoter in the pBI121 vector. CaMV35S The promoter was used to obtain the recombinant plasmid p397b-pBI121. The pBI121 vector itself contains... GUS The gene was used as the target gene, and the effects of recombinant plasmids containing different promoters on the infection of isolated pear organs were detected. GUS Gene expression levels, confirming PcMIR397b The promoter has significant advantages: in multiple tissues of pear, such as stems and leaves, its driving force... GUS Gene expression efficiency is not only significantly higher than that of heterologous genes. CaMV35S The promoter is also superior to that of the pear itself. pMIR397a promoter, pUBQ promoter, pU6 The promoter exhibits stronger expression-driven capabilities and broad organizational applicability. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a plasmid map of the pear transient transformation vector p397b-pBI121 in Example 1 of the present invention; Figure 2 This is a comparison image of GUS staining and chlorophyll removal of different pear plants in Example 1 of the present invention. Figure 2 a represents the pear plant CaMV35S. Figure 2 b represents the pear plant pUBQ. Figure 2 c represents pear plant pU6. Figure 2 d represents pear plant p397b; Figure 3 The statistical results of GUS activity of different pear in vitro plants in Example 1 of the present invention; Figure 4 This is the statistical result of the relative expression level of the GUS gene in different pear detached plants in Example 1 of the present invention; Figure 5 This is a comparison image of different pear detached leaves after GUS staining and chlorophyll removal in Example 2 of the present invention. Figure 5 a represents CaMV35S from pear leaves. Figure 5 b represents pear leaf pUBQ. Figure 5 c represents pear leaf PU6. Figure 5 d represents pear leaf p397b; Figure 6 The statistical results of GUS activity of different pear detached leaves in Example 2 of the present invention are shown. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0029] Unless otherwise specified, the raw materials and reagents used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0030] Example 1 This invention provides a pear transient transformation vector. This embodiment uses the pBI121 vector as the backbone plasmid to illustrate the construction method of the pear transient transformation vector. The backbone plasmid can also be other commonly used plant expression vectors in the art, such as the pCAMBIA series vectors, pBI221 vector, pBI121 vector, pBI121-EGFP vector, pBI121-mCherry, or pUBI10-GUS vector, etc. The specific construction method of the pear transient transformation vector is as follows: (1) Pear Source PcMIR397b promoter amplification Liyuan PcMIR397 Two transcripts, a and b, were detected in the regulatory region corresponding to the promoter. Preliminary experiments verified that pear-derived... PcMIR397 The α and β transcript gene sequences of the promoter were used to replace the CaMV35S promoter sequence in the pBI121 vector, resulting in recombinant plasmids p397a-pBI121 and p397b-pBI121, respectively. Subsequently, the two recombinant plasmids and the pBI121 vector were transformed into Agrobacterium using a freeze-thaw method, yielding recombinant strains p397a-pBI121, p397b-pBI121, and pBI121, respectively.

[0031] Under the same conditions, the three recombinant strains were used to infect pear detached plants. After cultivation, the expression level of the GUS gene in the pear detached plants infected with different recombinant strains was measured. The experiment showed that the expression level of the GUS gene in the p397a-pBI121 infection group was significantly lower than that in the pBI121 infection group, and the expression level of the GUS gene in the pBI121 infection group was significantly lower than that in the p397b-pBI121 infection group. Therefore, subsequent selection of pear-derived pears for further research is necessary. PcMIR397 The promoter's b transcript (i.e. PcMIR397b Further research will be conducted on the promoter.

[0032] This invention uses the 'Comfort' pear plant as material, extracts its genomic DNA and performs PCR amplification, and finally determines the origin of this pear. PcMIR397b The nucleotide sequence of the promoter is shown in SEQ ID NO.1.

[0033] Based on the nucleotide sequence shown in SEQ ID NO.1, primer pair pPcMIR397b-F / pPcMIR397b-R was designed; using pear 'Comfort' DNA as a template, pear-derived DNA was amplified by PCR. PcMIR397b Promoter gene. After PCR amplification, the amplification products were detected by agarose gel electrophoresis to verify the bands. Subsequently, the target band was recovered and sequenced for verification. The sequences of the amplification primer pairs are shown in Table 1.

[0034] The PCR reagents used in the experiment were purchased from Novizan Biotechnology Co., Ltd. The amplification reaction conditions were set as follows: 95℃ pre-denaturation for 3 min; followed by 34 cycles, each cycle including: 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min; and finally 72℃ final extension for 3 min.

[0035] Table 1

[0036] The nucleotide sequence shown in SEQ ID NO.1 is as follows:

[0037] (2) Construction of pear transient transformation carrier Using the pBI121 vector as a backbone plasmid, the gene sequence of the pear-derived PcMIR397b promoter, located between the double restriction sites on the pBI121 vector, was replaced by double digestion with HindIII and SmaI. CaMV35S Promoter gene sequence, using T4 DNA ligase to connect pear-derived... PcMIR397b The promoter gene sequence was inserted into the 5' end of the GUS gene. Positive clones in the recombinant plasmid were screened and identified using primers M13-R (sequence: CAGGAAACAGCTATGAC) and PcMIR397b-R (sequence shown in Table 1), yielding the modified pear-derived gene. PcMIR397b The recombinant plasmid p397b-pBI121, which is the promoter of the pear transient transformation vector, is shown in the following plasmid map. Figure 1 As shown.

[0038] Example 2 Taking the pear transient transformation vector p397b-pBI121 provided in Example 1 as an example, this invention provides a method for transient transformation of pear plants, comprising the following steps: S1. Following the method described in Example 1, the pear transient transformation vector p397b-pBI121 was constructed and screened to obtain the pear transient transformation vector p397b-pBI121; S2. The pear transient transformation vector p397b-pBI121 was transformed into Agrobacterium GV3101 by freeze-thaw method to obtain the recombinant strain p397b-pBI121; S3. After graded expansion culture and induction culture, the recombinant strain p397b-pBI121 was mixed with pear explants and then subjected to vacuum infiltration infection to obtain pear explants infected with the recombinant strain, as detailed below: S31. The recombinant strain p397b-pBI121 bacterial culture (OD) 600 =0.8) 6 μL was inoculated into 3 mL of YEB medium containing 50 mg / L rifampin (Rif) and 100 mg / L kanamycin (Kan), and cultured in a shaker at 28℃ and 180 rpm for 16 h to obtain the primary expansion medium; S32. Take 3 mL of the primary expansion medium and inoculate it into 30 mL of YEB medium containing 50 mg / L Rif, 100 mg / L Kan, 10 mM MES and 20 μM acetylsyringone (AS). Incubate in a shaker at 28℃ and 180 rpm for 5 h to obtain the secondary expansion medium. S33. Transfer the above secondary expansion broth to a 50 mL centrifuge tube, centrifuge at 4500 rpm for 10 min to collect the bacterial cells, resuspend the bacterial cells in induction medium, and adjust the OD of the bacterial suspension. 600The culture was incubated at 28℃ for 3 hours to obtain the induced recombinant bacterial solution. The induction solution contained 10 mM MgCl2, 10 mM MES and 150 μM AS, with the remainder being water and a pH of 5.6. S34. Place the pear plants that have been subcultured for 3 weeks into the above-mentioned recombinant bacterial solution after induction, add 0.01% silwet L-77 and mix well. Place the solution in a vacuum pump and perform vacuum permeation infection twice at a vacuum degree of -0.09 MPa, each time for 5 minutes. After the infection is completed, remove the plants infected with the recombinant strain, and use filter paper to absorb the excess bacterial solution to obtain the pear plants infected with the recombinant strain, which are recorded as pear plant p397b.

[0039] S4. Pear plants p397b were placed in MS solid medium and cultured in the dark at 25°C for 12 hours, followed by photoperiod culture at 25°C for 48 hours. The expression of the GUS gene was then measured. The photoperiod was set to 18 hours of light and 6 hours of darkness.

[0040] Example 3 Taking the pear transient transformation vector p397b-pBI121 provided in Example 1 as an example, this invention provides a method for transient transformation of pear plants. The method for transient transformation of pear plants is basically the same as that in Example 2, except that: (1) in step S34, “tissue culture seedling leaves” are used instead of “pear in vitro plants” and the name after infection is changed accordingly; (2) in step S4, after 12 hours of dark culture at 25℃, photoperiodic culture is performed for 2 days, and then the expression of GUS gene is measured.

[0041] Steps S34 and S4 are as follows: S34. Place the pear tissue culture seedling leaves that have been subcultured for 3 weeks into the above-mentioned recombinant bacterial solution after induction, add 0.01% silwet L-77 and mix well. Place the solution in a vacuum pump and perform vacuum permeation infection twice at a vacuum degree of -0.09 MPa, each time for 5 minutes. After the infection is completed, remove the leaves infected with the recombinant strain, and use filter paper to absorb the excess bacterial solution to obtain the pear detached leaves infected with the recombinant strain, which are recorded as pear leaf p397b.

[0042] S4. The pear leaves p397b were placed in MS solid medium and cultured in the dark at 25°C for 12 hours, followed by photoperiod culture at 25°C for 48 hours. The expression of the GUS gene was then measured. The photoperiod was set to 18 hours of light and 6 hours of darkness.

[0043] Comparative Example 1 This comparative example provides a method for constructing a pear transient transformation vector, which is basically the same as that in Example 1, except that the pear source is amplified. pUBQAfter the promoter gene sequence, pear source pUBQ The promoter gene sequence of the pBI121 vector was replaced. CaMV35S The promoter gene sequence was used to obtain the pear transient transformation vector pUBQ-pBI121. The pear-derived gene was used in this process. pUBQ The promoter gene sequence is shown in SEQ ID NO.2.

[0044] Comparative Example 2 This comparative example provides a method for constructing a pear transient transformation vector, which is basically the same as that in Example 1, except that the pear source is amplified. pU6 After the promoter gene sequence, pear source pU6 The promoter gene sequence of the pBI121 vector was replaced. CaMV35S The promoter gene sequence was obtained, ultimately leading to the pear transient transformation vector pU6-pBI121. Among them, the pear source... pU6 The promoter gene sequence is shown in SEQ ID NO.3.

[0045] Example of effect 1 In this invention, the pear transient transformation vectors pUBQ-pBI121, pear transient transformation vector pU6-pBI121, and pBI121 vector, constructed in a comparative manner, were transformed into Agrobacterium GV3101 according to the method described in step S2 of Example 2, thereby constructing recombinant strains pUBQ-pBI121, pU6-pBI121, and pBI121, respectively. Further, the above three recombinant strains were used to prepare pear in vitro plants infected with the recombinant strains according to the method described in step S3 of Example 2, respectively, and were designated as pear plant pUBQ, pear plant pU6, and pear plant CaMV35S, respectively. Similar to Example 2, each recombinant strain transformed at least 9 pear in vitro plants.

[0046] GUS staining was performed on pear plants infected with different recombinant strains in vitro, or their GUS activity and GUS gene expression levels were measured. The specific methods are as follows: After culturing according to the method described in step S4 of Example 2, pear plants p397b, pUBQ, pU6, and CaMV35S were stained with GUS using a GUS staining kit (Coolaber, China) according to its instructions. After staining, each plant was immersed in 75% ethanol for 1 hour to remove chlorophyll. The criteria for judging the staining results were: a white plant indicated no GUS expression or extremely low expression; the deeper the color (blue), the higher the GUS expression level.

[0047] Equal weights of different plant materials were taken, and the GUS activity of pear plant p397b, pear plant pUBQ, pear plant pU6 and pear plant CaMV35S was measured according to the instructions using a GUS activity assay kit (FCNCS, China). Three plants were selected for each group.

[0048] To determine GUS gene expression levels, total RNA was extracted from different plants, and cDNA was synthesized via reverse transcription. The cDNA was then detected using RT-qPCR. The specific RT-qPCR method is as follows: Using 1.5 μg of total RNA as a template, cDNA was obtained through reverse transcription using the HiScript Q RTSuperMix for qPCR (+gDNA wiper) kit (Novizan, Nanjing). qPCR was then performed using the ChamQ Universal SYBR qPCR Master Mix kit (Novizan, Nanjing), with PcActin gene used as an internal control to correct for differences between samples. The gene sequences of the primer pairs pear-actin-F / R and GUS-F / R required for amplification are shown in Table 2.

[0049] Table 2

[0050] The PCR amplification program was as follows: pre-denaturation at 95℃ for 30 seconds; followed by 40 cycles, each cycle consisting of: denaturation at 95℃ for 30 seconds, annealing at 60℃ for 30 seconds, extension at 72℃ for 15 seconds, and a final extension at 72℃ for 3 minutes. After amplification, fluorescence signals were collected starting at 55℃, with a 10-second pause for each 0.5℃ increase in temperature, until the melting curve was collected at 95℃. Three technical replicates were set up for each sample, for a total of three biological replicates.

[0051] In this invention, the expression level of the GUS gene is used as 2 -△△Ct The relative quantitative method was used for calculation, and the t-test was performed using the software Sigmaplot to analyze the significance of the differences (P<0.05).

[0052] Comparison of GUS staining and chlorophyll removal of different pear plants in vitro is shown in the following figures. Figure 2 As shown, where Figure 2 a represents the pear plant CaMV35S. Figure 2 b represents the pear plant pUBQ. Figure 2 c represents pear plant pU6. Figure 2 d represents pear plant p397b. The driving ability of different promoters on the GUS gene was evaluated by analyzing GUS staining patterns in different plants.

[0053] Depend on Figure 2It was found that, compared to the light blue leaves of pear plants CaMV35S, pUBQ, and pU6, the blue color of pear plant p397b leaves was deeper and more evenly distributed. In terms of stem appearance, the stems of pear plants CaMV35S, pUBQ, and pU6 were light green, while the stems of pear plant p397b were uniformly blue-green. In summary, the deeper and more evenly distributed blue staining of the stems and leaves of pear plant p397b suggests that, compared to the CaMV35S, pUBQ, and pU6 promoters, the p397b promoter has a stronger driving effect on GUS gene expression in the stems and leaves of isolated pear plants.

[0054] Statistical results of GUS activity in different pear in vitro plants are as follows: Figure 3 As shown. By Figure 3 It can be seen that the pear plant p397b has the highest GUS activity, reaching 246 nmol·mg. -1 ·min -1 The GUS activity in p397b of the pear plant was significantly higher than that in pU6 and CaMV35S of the pear plant, and significantly higher than that in pUBQ of the pear plant.

[0055] The statistical results of the relative expression levels of the GUS gene in different pear detached plants are as follows: Figure 4 As shown. By Figure 4 It can be seen that the expression level of the GUS gene is the highest in pear plant p397b, which is significantly higher than that in pear plant pU6 and pear plant CaMV35S.

[0056] Example 2 In this invention, the recombinant strains pUBQ-pBI121, pU6-pBI121, and pBI121 constructed in Example 1 were used to prepare pear leaves infected with the recombinant strains according to the method described in step S3 of Example 3. These leaves were designated as pear leaf pUBQ, pear leaf pU6, and pear leaf CaMV35S, respectively. Similar to Example 3, each recombinant strain was used to transform at least 20 pear leaves.

[0057] After culturing according to the method described in step S4 of Example 3, pear leaves p397b, pUBQ, pU6, and CaMV35S were stained with GUS using a GUS staining kit (Coolaber, China). After staining, each leaf was immersed in 75% alcohol for 1 hour to remove chlorophyll.

[0058] The GUS activity of pear leaf p397b, pUBQ, pU6 and CaMV35S was determined according to the instructions using a GUS activity assay kit (FCNCS, China), with three leaves selected for each group.

[0059] Comparison images of GUS staining and chlorophyll removal of different pear detached leaves are shown below. Figure 5 As shown, where Figure 5 a represents CaMV35S from pear leaves. Figure 5 b represents pear leaf pUBQ. Figure 5 c represents pear leaf PU6. Figure 5 d represents pear leaf p397b. (From...) Figure 5 It can be seen that the GUS staining of pear leaves CaMV35S and pU6 is relatively light and unevenly distributed; in contrast, the staining of pUBQ pear leaves is darker and more evenly distributed, while the staining of p397b pear leaves is more intense than that of pUBQ pear leaves.

[0060] Statistical results of GUS activity in different pear detached leaves are as follows: Figure 6 As shown. By Figure 6 It can be seen that the GUS activity in p397b of pear leaves is significantly higher than that in pU6 and CaMV35S of pear leaves, and significantly higher than that in pUBQ of pear leaves.

[0061] In summary, among detached pear plants and detached pear leaves, pear-derived pear... PcMIR397b The promoter has significant advantages in driving GUS gene expression. Firstly, pear-derived... PcMIR397b Promoter-driven GUS staining is deeper blue and more evenly distributed, visually demonstrating stronger expression ability; secondly, GUS activity and relative gene expression are the highest, and there are significant or extremely significant differences compared with other promoters, confirming that its expression efficiency is superior.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Pyrus pyrifolia PcMIR397b Use of the promoter in constructing a transient transformation vector for Pyrus pyrifolia.

2. The pear source of claim 1 PcMIR397b Use of the promoter in the construction of a transient transformation vector for pear, characterized in that, The pear source PcMIR397b The nucleotide sequence of the promoter is shown in SEQ ID NO.

1.

3. A pear transient transformation vector, characterized in that, The pear transient transformation vector is a vector comprising the pear source as claimed in claim 1 or 2 PcMIR397b Recombinant vector of the promoter gene; The recombinant vector comprises a target gene.

4. The pear transformation vector according to claim 3, wherein The recombinant vector takes a plant expression vector as a backbone; The plant expression vector comprises at least one of a pCAMBIA series vector, a pBI221 vector, a pBI121 vector, a pBI121-EGFP vector, a pBI121-mCherry vector, or a pUBI10-GUS vector.

5. The pear transformation vector according to claim 3, wherein The target gene comprises a reporter gene or an exogenous gene in the vector backbone.

6. A method for the transient transformation of a pear plant, characterized in that, The method comprises the following steps: S1. Constructing the pear transient transformation vector according to any one of claims 3-5; S2. Transforming the pear transient transformation vector into Agrobacterium to obtain a recombinant strain; S3. After the recombinant strain is subjected to a subculture and an induction culture, the recombinant strain is mixed with pear explants, vacuum infiltration is performed after a surfactant is added, and pear explants infected with the recombinant strain are obtained; S4. After the pear explants infected with the recombinant strain are cultured, the expression of the target gene is determined.

7. The method of transient transformation of a pear plant as claimed in claim 6, wherein, The pear explants comprise pear in vitro plants or leaf pieces of tissue culture seedlings.

8. The method of transient transformation of a pear plant as claimed in claim 6, wherein, In step S4, the culture comprises dark culture and / or photoperiod culture; The dark culture is performed at a temperature of 23-27°C for 6-18 hours; and / or The photoperiod is set to 10-18 hours of light and 6-14 hours of darkness; and / or The photoperiod culture is performed at a temperature of 23-27°C for 24-72 hours.

9. The method of transient transformation of a pear plant according to any one of claims 6 to 8, wherein the plant is a pear plant of the species Pyrus communis. 5 The method comprises the following steps: S1. With pBI121 vector as the skeleton, the nucleotide sequence as shown in SEQ ID NO. 1 is replaced with the gene sequence of the CaMV35S promoter in the pBI121 vector to obtain the pear transient transformation vector p397b-pBI121. PcMIR397b The gene sequence of the promoter replaces the gene sequence of the CaMV35S promoter in the pBI121 vector to obtain the pear transient transformation vector p397b-pBI121. S2. Transforming the pear transient transformation vector p397b-pBI121 into Agrobacterium to obtain a recombinant strain; S3. After the recombinant strain is subjected to a subculture and an induction culture, the recombinant strain is mixed with pear in vitro plants or leaf pieces of tissue culture seedlings, vacuum infiltration is performed after a surfactant is added, and pear explants infected with the recombinant strain are obtained; S4. After the pear explants infected with the recombinant strain are subjected to dark culture and photoperiod culture, the expression of the GUS gene is determined.

10. The method of transient transformation of a pear plant as claimed in claim 9, wherein, The method for constructing the pear explants infected with the recombinant strain comprises the following steps: S31. Inoculating the recombinant strain into YEB medium containing rifampicin and kanamycin, and culturing at 25-30°C and 160-200 rpm for 14-18 hours to obtain a first subculture solution; S32. Inoculating the first subculture solution into YEB medium containing rifampicin, kanamycin, MES buffer, and acetyl-piperitone, and culturing at 25-30°C and 160-200 rpm for 4-6 hours to obtain a second subculture solution; S33. Collecting bacteria in the second subculture solution, resuspending the bacteria by adding an induction solution, and standing and culturing at 25-30°C for 2-4 hours to obtain an induced recombinant bacterial solution; S34. Mixing the induced recombinant bacterial solution with pear in vitro plants or leaf pieces of tissue culture seedlings after subculture, adding a silicone surfactant, and performing vacuum infiltration to obtain pear explants infected with the recombinant strain.

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